Prediction of Molar Extinction Coefficients of Proteins and Peptides Using UV Absorption of the Constituent Amino Acids at 214 nm To Enable Quantitative Reverse Phase High-Performance Liquid Chromatography-Mass Spectrometry Analysis

B.J.H. Kuipers, H. Gruppen

Research output: Contribution to journalArticleAcademicpeer-review

203 Citations (Scopus)

Abstract

The molar extinction coefficients of 20 amino acids and the peptide bond were measured at 214 nm in the presence of acetonitrile and formic acid to enable quantitative comparison of peptides eluting from reversed-phase high-performance liquid chromatography, once identified with mass spectrometry (RP-HPLC-MS). The peptide bond has a molar extinction coefficient of 923 M-1 cm-1. Tryptophan has a molar extinction coefficient that is ~30 times higher than that of the peptide bond, whereas the molar extinction coefficients of phenylalanine, tyrosine, and histidine are ~six times higher than that of the peptide bond. Proline, as an individual amino acid, has a negligible molar extinction coefficient. However, when present in the peptide chain (except at the N terminus), it absorbs ~three times more than a peptide bond. Methionine has a similar molar extinction coefficient as the peptide bond, while all other amino acids have much lower molar extinction coefficients. The predictability of the molar extinction coefficients of proteins and peptides, calculated by the amino acid composition and the number of peptide bonds present, was validated using several proteins and peptides. Most of the measured and calculated molar extinction coefficients were in good agreement, which shows that it is possible to compare peptides analyzed by RP-HPLC-MS in a quantitative way. This method enables a quantitative analysis of all peptides present in hydrolysates once identified with RP-HPLC-MS. Keywords: Molar extinction coefficient; molar absorption coefficient; UV absorbance; proteins; peptides; amino acids
Original languageEnglish
Pages (from-to)5445-5451
JournalJournal of Agricultural and Food Chemistry
Volume55
Issue number14
DOIs
Publication statusPublished - 2007

Keywords

  • beta-lactoglobulin hydrolysis
  • enzyme inhibitory peptides
  • bioactive peptides
  • spectra
  • identification
  • polypeptide
  • conglycinin

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